Enzymatic synthesis of optically active hydroxamic acids and their conversion to optically active primary amines by a lossen rearrangement

ABSTRACT

A method is described for the preparation of optically active hydroxamic acids of the general formula                    
     wherein R 1 , R 2  and R 3  are different and are a cyclic or linear, aliphatic or aromatic, substituted or unsubstituted hydrocarbon radical which can optionally contain heteroatoms, said method being characterized in that a racemate of chiral amides, carboxylic acid esters or carboxylic acids of the general formula                    
     wherein R 1 , R 2  and R 3  are as defined above and X is —NH 2 , —OR or —OH, R being any organic radical, 
     is reacted with hydroxylamine, NH 2 OH, in the presence of an acyltransferase, and the optically active hydroxamic acid (I) formed is then separated from the unconverted enantiomer of general formula (II). The resulting optically active hydroxamic acid can be converted to the corresponding optically active primary amines by a Lossen rearrangement.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the enzymatic synthesis of optically activehydroxamic acids and to their conversion to optically active primaryamines by a Lossen rearrangement.

2. Background of the Prior Art

Hydroxamic acids are compounds of great pharmaceutical interest.Hydroxamic acid derivatives exist which have antibacterial andfungicidal properties (Duda et al., 1965; Hase et al., 1971).Alkylaminopropionohydroxamic acids exhibit hypotensive properties(Coutts et al., 1971). Hypocholesterolaemic actions have also beendemonstrated for hydroxamic acids (Ludwig et al., 1967).p-Butoxyphenylacetohydroxamic acid possesses an anti-inflammatory action(Dell et al., 1971) and is used in human medicine. Some hydroxamic acidshave been studied for their efficacy against malaria (Hynes, 1970; Hynes& Hack, 1972).

Enantiomerically pure hydroxamic acids, however, are of particularimportance. Their pharmacological activity is higher than that of theracemates. Furthermore, via the Lossen rearrangement, they also open upa new route to chiral primary amines. This class of substances ispharmacologically of great importance as well. Thus chiral β-aminoalcohols, for example, are used in large amounts as β-adrenoceptorantagonists, abbreviated to β-blockers.

However, the preparation of enantiomerically pure hydroxamic acids bythe conventional methods of organic chemistry is expensive. It normallyrequires a racemate separation or the use of metal-organic catalysts,but the latter are generally unsuitable for the preparation of drugs.Such methods are described for example in DE-PS 2 400 531, EP-A-203 379and EP-A-268 215.

SUMMARY OF THE INVENTION

The object of the invention is therefore to provide a novel method ofpreparing optically active hydroxamic acids which is not associated withthe above-mentioned problems.

Surprisingly it has now been found that amides, carboxylic acid estersand carboxylic acids which have a centre of chirality on the α-carbonatom can be enantioselectively converted to optically active hydroxamicacids by acyltransferases in the presence of hydroxylamine.

The invention therefore provides a method of preparing optically activehydroxamic acids of the general formula

wherein R¹, R² and R³ are different and are a cyclic or linear,aliphatic or aromatic, substituted or unsubstituted hydrocarbon radicalwhich can optionally contain heteroatoms, said method beingcharacterized in that a racemate of chiral amides, carboxylic acidesters or carboxylic acids of the general formula

wherein R¹, R² and R³ are as defined above and X is —NH₂, —OR or —OH, Rbeing any organic radical, is reacted with hydroxylamine, NH₂OH, in thepresence of an acyltransferase, and the optically active hydroxamic acid(I) formed is then separated from the unconverted enantiomer of generalformula (II).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph plotting, against the reaction time in minutes, theconcentration in mM of phenylacetohydroxamic acid (▾), acetohydroxamicacid (♦) and phenylacetic acid (▪) formed in a representative process ofthe invention reacting acetamide and phenylacetamide with hydroxylaminein the presence of an acyltransferase from quiescent cells ofRhodococcus erythropolis MP50;

FIG. 2 is a graph plotting, against the reaction time in minutes, theconcentration in mM of phenylacetohydroxamic acid (▾), acetohydroxamicacid (♦) and phenylacetic acid (▪) formed in a representative process ofthe invention reacting acetamide and phenylacetamide with hydroxylaminein the presence of an acyltransferase from a crude extract ofRhodococcus erythropolis MP50; and

FIG. 3 is a graph depicting the formation of 2-phenylpropionohydroxamicacid () in mM for the reaction of (R,S)-2-phenylpropionamide (▴) withhydroxylamine in the presence of acyltransferase recovered fromRhodococcus erythropolis MP50, with racemic 2-phenylpropionamide (◯) asthe model substrate.

DETAILED DESCRIPTION OF THE INVENTION, PREFERRED EMBODIMENTS AND BESTMODE

Suitable acyltransferases, i.e. enzymes which transfer the acyl radicalto the hydroxylamine, are known per se. They can be isolated for examplefrom the following microorganisms: Mycobacteriaceae, Mycobacteriumsmegmatis, Pseudomonas aeruginosa, Arthrobacter, Aspergillus nidulans,Bradyrhizobium japonicum, Brevibacterium sp. R312, Methylophilusmethylotrophus, Pseudomonas chlororaphis, Pseudomonas fluorescens,Rhodococcus rhodochrous Ji and Rhodococcus erythropolis MP50.

The microorganism Rhodococcus erythropolis MP50 was deposited with DSMZ(Deutsche Samnilung von Mikroorganismen und Zellkulturen GmbH)Mascheroder Weg 1b, D-38124 Braunschweig, Germany, on Jan. 16, 1995, andassigned DSMZ Accession No. 9675.

The acyltransferase isolated from Rhodococcus erythropolis MP50 is foundto be very particularly suitable. Of all the amidases studied, this onehad the highest specific activities and the broadest substrate spectrum.Amidase and acyltransferase activity can be found in the same enzyme.The amidase activity is high too, but the acyltransferase activity ismarkedly higher, probably because hydroxylamine is a better acyl radicalacceptor than water. Thus, by using this special acyltransferase, i.e.the acyltransferase isolated from Rhodococcus erythropolis MP50,hydrolysis of the amides and esters used as starting materials can beextensively avoided.

The method according to the invention can be carried out using either acrude extract of the acyltransferase from said microorganisms or apurified form. Using the crude extract has the advantage that the enzymepurification step can be dispensed with. For special applications,however, it is recommended to use purified acyltransferase. Inparticular, it is found that the catalytic activity of the purifiedacyltransferase is higher than that of the crude extract, so chemicallyinert starting materials are preferably reacted with the purifiedacyltransferase.

The recovery and purification of the acyltransferase from Rhodococcuserythropolis MP50 is described in “Purification and properties of anamidase from Rhodococcus erythropolis MP50 which enantioselectivelyhydrolyzes 2-aryilpropionamides”, B. Hirrlinger, A. Stolz & H. -J.Knackmuss, Journal of Bacteriology, 1996, vol. 178(12), pp. 3501-3507.

The cells or the purified enzyme can be used directly or in immobilizedform. Immobilization enables the biocatalyst to have multiple uses andsimplifies the working-up of the reaction mixture. Within the frameworkof the present invention, whole cells or purified enzyme are immobilizedby methods known per se. Whole cells are immobilized e.g. with alginate,κ-carrageenan, polyurethane or polyacrylamides (I. Chibata, T. Tosa & T.Sato, 1983, Immobilized Cells in Preparation of Fine Chemicals, AdvancesIn Biotechnological Processes (A. R. Liss, editor), volume 1, pp.203-222.

Purified enzyme is immobilized either by adsorption onto suitablesupports (Célite, cellulose), by ionic binding to an ion exchanger resin(DEAE-cellulose, Sephadex), by covalent bonding to a carrier (porousglass beads, cellulose, dextran, agarose) or by inclusion in a gel(alginate, agar, polyacrylamide) (O. R. Zaborsky, 1973, ImmobilizedEnzymes, CRC Press, Cleveland, Ohio; M. D. Trevan, 1980, ImmobilizedEnzymes: Introduction and Applications in Biotechnology, Wiley, NewYork; W. Hartmeier, 1986, Immobilisierte Biokatalysatoren (ImmobilizedBiocatalysts), Springer, Berlin).

As the method according to the invention is an enzyme-catalyzedreaction, the pH of the reaction medium is to be taken intoconsideration. It should generally be in the pH range 5.5 to 9 andpreferably in the pH range 6.5 to 7.5. Suitable buffer systems, such assodium phosphate buffer and Tris/HCl buffer, can be used for thispurpose.

In principle, it is possible to use any amides, carboxylic acid estersand carboxylic acids as starting materials for the preparation of thedesired hydroxamic acids, provided that the carbon atom adjacent to thecarbonyl carbon, i.e. the α-carbon atom, is chiral. This means that thesubstituents present on this carbon atom have to be different. Thesesubstituents can be any cyclic or linear, aliphatic or aromatic,substituted or unsubstituted hydrocarbon radicals and can optionallycontain heteroatoms such as oxygen, sulfur, nitrogen, phosphorus, etc.

Substituents such as methyl, ethyl, n-pentyl, isopropyl, cyclohexyl,phenyl and naphthyl are found to be particularly suitable.

Particularly suitable starting materials are thereforealkylaminopropionic acid, p-butoxyphenylacetic acid, 2-phenylpropionicacid, 2- (6-methoxy-2-naphthyl) propionic acid (naproxen),2-(3′-benzoylphenyl)propionic acid (ketoprofen), 2-methylbutanoic acid,2-(1-naphthyl)propionic acid and their esters or amides.

In general, it is preferable to use the amides of the compounds coveredby general formula (I), being more reactive than the correspondingcarboxylic acids or carboxylic acid esters.

The concentration of the reactants in the reaction medium is notparticularly critical. The compounds of formula (I) are generally usedin a concentration of 0.5 to 0.001 mol./l and preferably in aconcentration of 0.1 mol./l. The concentration of the hydroxylamine isgenerally 1.0 to 0.1 mol./l and preferably 0.5 to 0.2 mol./l. The ratioof the compounds of formula (I) to hydroxylamine is therefore generally1:10 to 1:2 and preferably 1:5. The activity of the acyltransferase isgenerally 3.5 to 37 units/mg protein and preferably 13 units/mg protein(with 2-phenylpropionamide as substrate).

The essential advantage of the method according to the invention is tobe regarded as the fact that the acyltransferase selectively catalyzesthe conversion of only one enantiomer of the racemate of opticallyactive amides, carboxylic acid esters or carboxylic acids, used asstarting material, with hydroxylamine to the corresponding hydroxamicacid, while its mirror image remains unchanged in the reaction. Thereaction mixture initially obtained directly after the conversiontherefore contains the optically pure hydroxamic acid together with theunconverted enantiomer of the starting material. These two componentscan easily be separated from one another by shaking the reaction mixturewith ethyl acetate, diethyl ether or methylene chloride to extract it,the general procedure being as follows:

The aqueous reaction solution is adjusted to pH 10 with sodium hydroxidesolution. The amide is extracted with one of the above-mentioned organicsolvents. The aqueous phase is then adjusted to pH 2 with hydrochloricacid. The hydroxamic acid is then extracted into the organic phase byshaking and is thereby isolated.

The optically active hydroxamic acid recovered in this way can then beconverted in a manner known per se, by a Lossen rearrangement, to thecorresponding optically active and enantiomerically pure primary amine.The invention therefore also provides a method of preparing opticallyactive primary amines of the general formula

wherein R¹, R² and R³ are as defined above, in which method theoptically active hydroxamic acid of general formula (I), obtained asdescribed above, is O-acylated, the O-acylated hydroxamic acid isconverted to the corresponding isocyanate by heating or by treatmentwith bases in aprotic solvents, and the isocyanate is reacted with waterto give the amine of general formula (III) with the elimination of CO₂.

The Lossen rearrangement therefore proceeds according to the followingreaction scheme:

Suitable bases are compounds like NaOH, KOH, NaNH₂ and Na₂CO₃ the natureof the bases used depending on the structure of the desired hydroxamicacid .

Examples of convenient aprotic solvents are toluene, benzene or xylene.To decompose the O-acylated hydroxamic acid as quantitatively and asrapidly as possible, temperatures of 100° C. to 150° C., preferably 100°C., are generally convenient.

As the absolute configuration of the α-carbon atom of the opticallyactive hydroxamic acid used as starting material is retained in theLossen rearrangement (cf. Campbell & Kenyon, 1946), the primary aminesobtained as products of the Lossen rearrangement are also opticallyactive.

However, in the method according to the invention for the preparation ofoptically active primary amines, it is not necessary to remove theunconverted enantiomer of the amides, carboxylic acid esters orcarboxylic acids used in the preparation of the optically activehydroxamic acids, after the first enzyme-catalyzed reaction, i.e. it isnot necessary to use the pure optically active hydroxamic acid. Thereaction can also be carried out in such a way that the racemate of thechiral amides, carboxylic acid esters or carboxylic acids is firstreacted with hydroxylamine under enzyme catalysis, and the resultingreaction mixture, which contains the unconverted enantiomer of thestarting material together with the optically active hydroxamic acid, isreacted with a carbodiimide under protonic catalysis to form anisocyanate and a corresponding urea derivative as intermediates. Theisocyanate then reacts immediately with the water contained in thereaction system to give a carbamic acid, which in turn decomposes tocarbon dioxide and the desired optically active primary amine. The aminecan then easily be separated from the unconverted enantiomer of thestarting material.

The reaction with carbodiimide has the advantage that the opticallyactive hydroxamic acid obtained in an aqueous reaction system canimmediately be processed further, thereby obviating the need for apurification step. The derivatization with a carbodiimide is necessarybecause the conventional chemical methods of O-acylating hydroxamic acidcan only be carried out in aprotic solvents, which are inconvenient forenzyme-catalyzed reactions.

Examples of suitable carbodiimides are1-benzyl-3-(3′-dimethylaminopropyl)carbodiimide,1-cyclohexyl-3-(2′-morpholinoethyl)carbodiimide and1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide.1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDC) is found to beparticularly convenient. This carbodiimide is described for example inSheehan, J. C., P. A. Cruickshank & G. L. Boshart, 1961, A convenientsynthesis of water-soluble carbodiimides, J. Org. Chem. 26, 2525-2528.

As an excess of hydroxylamine is generally used in the preparation ofthe optically active hydroxamic acid, it is convenient to remove theexcess hydroxylamine before the reaction mixture is reacted with thecarbodiimide. This is done by acidifying the reaction mixture to a pH inthe range 0 to 2 and preferably in the region of 1. The hydroxylaminethen readily decomposes to ammonium and nitrous oxide (cf. Holleman, A.F. & E. Wiberg, 1985, Lehrbuch der Anorganischen Chemie (Textbook ofInorganic Chemistry), edition 91-100, p. 591, Walter de Gruyter Verlag,Berlin, N.Y.).

After acidification, the reaction mixture is heated at a temperature inthe range 40° C. to 100° C., preferably at 80° C., until no furtherevolution of gas can be observed. A base, for example sodium hydroxidesolution, is then added to the reaction mixture in order to adjust thepH to a value of 4 to 6 and preferably to pH 5.0.

The derivatization with the carbodiimide is then-performed according tothe following general scheme:

wherein R¹ to R⁵ are as defined above.

If, for example, racemic 2-phenylpropionamide is used as the startingmaterial in the method according to the invention,S—(—)-phenylethylamine and R-2-phenylpropionamide are initially obtainedaccording to the following reaction scheme:

The desired optically active primary amine can easily be separated fromthe unconverted enantiomer of the starting material, a possibleprocedure being as follows: The aqueous reaction solution is acidifiedto pH 1 with hydrochloric acid and extracted with ethyl acetate, diethylether or methylene chloride. The amide is now in the organic phase. ThepH of the aqueous phase is then brought to 10 with sodium hydroxidesolution and the amine is extracted with the solvents mentioned.

This procedure affords optically active primary amines, such asS—(—)-phenylethylamine, S-2-aminobutane or S-1-(1-naphthyl)ethylamine,which would otherwise only be accessible by expensive racemateseparation.

The invention is illustrated in greater detail by means of the Examplesbelow.

The procedure for determining the hydroxamic acids was as follows:

As bidentate ligands, hydroxamic acids form stable, intensely colouredchelate complexes with iron(III) ions. These trishydroxamatoiron(III)complexes [Fe(RCONHO)₃] have an absorption maximum at 500 nm and have adeep red colour to the eye. This property is utilized for thequalitative determination (Buckles, R. E. & C. J. Thelen, 1950,Qualitative determination of carboxylic esters. Scope and limitations ofhydroxamic acid test. Anal. Chem. 22, 676-678) and quantitativedetermination (Brammar, W. J. & P. H. Clarke, 1964, Induction andrepression of Pseudomonas aeruginosa amidase. J. Gen. Microbiol. 37,307-319) of hydroxamic acids. The iron complex is obtained by mixing asolution containing hydroxamic acid with an iron(III) chloride solutionacidified with hydrochloric acid. The extinction is determined byspectrophotometry in a glass cell at 500 nm.

Iron(III) chloride solution (modified according to Hoare, D. G., A.Olson & D. E. Koshland JR., 1968. The reaction of hydroxamic acids withwater-soluble carbodiimides. A Lossen rearrangement. J. Am. Chem. Soc.90, 1638-1643):

FeCl₃.6H₂O 13.51 g conc. hydrochloric acid (12M) 3.33 ml H₂O ad 500 ml

The solution was filtered through a filter of pore size 0.45 μm toremove turbidity.

600 μl of the iron(III) chloride solution acidified with hydrochloricacid were pipetted into 300 μl of a sample containing hydroxamic acid[hydroxamic acid dissolved in Tris/HCl buffer (30 mM, pH 7.5)]. A deepred-coloured iron complex formed immediately. The extinction wasdetermined by spectrophotometry. Calibration lines were plotted withacetohydroxamic acid and 2-phenylpropionohydroxamic acid dissolved inTris/HCl buffer (30 mM, pH 7.5). These showed a linear dependencebetween extinction and hydroxamate concentration in the range 0.0 to 1.5mM hydroxamic acid. The molar extinction coefficients (ε_(λmax)500 nm)determined for acetohydroxamic acid, phenylacetohydroxamic acid and2-phenylpropionohydroxamic acid were 3500 lmol.⁻¹ cm⁻¹ and 4500 lmol.⁻¹cm⁻¹ respectively.

Enzymatic activities were determined both by means of HPLC and byphotometric methods. The photometric measurements were made at roomtemperature in 1 ml glass cells with a path length of 1 cm.

The specific activity is indicated in enzyme units per mg of protein(U/mg). One unit is the enzymatic activity which catalyzes theconversion of 1 μmol. of substrate or the formation of 1 μmol. ofproduct in one minute.

EXAMPLE 1

Cells of Rhodococcus erythropolis MPSO were grown in ammonium-freemineral medium with succinate (10 mM) as the carbon and energy source,ketoprofenamide (1 mM) as the nitrogen source and 3% of complex medium(NB). At the end of the exponential growth phase, the cells wereharvested by centrifugation, washed once in Tris/HCl buffer (30 mM, pH7.5) and resuspended in the same buffer to give an optical density(OD₅₄₆nm) of 16.0. 2.75 ml of Tris/HCl buffer (30 mM, pH 7.5) wereplaced in each of two 25 ml Erlenmeyer flasks with baffles and 54 mg ofphenylacetamide (PAA) or 23.6 mg of acetamide were added. The flaskswere then incubated at 30° C. in a vibrating water bath until the PAA orthe acetamide had completely dissolved. 1 ml of a freshly preparedhydroxylamine hydrochloride solution (2 M), freshly neutralized withNaOH (10 M), was added to each flask. The reaction was started by theaddition of 0.25 ml of the cellular suspension to each flask. The totalvolume was 4 ml. The optical density (OD₅₄₆nm) in the experiment was1.0. The batch contained 0.1 M amide and 0.5 M hydroxylamine. 0.3 mlaliquots of the cellular suspensions were withdrawn at intervals of 2min and pipetted into 600 μl of an iron(III) chloride solution acidifiedwith hydrochloric acid. The cells were removed by centrifugation and thesupernatant was diluted again in a ratio of 1 to 10 with iron(III)chloride solution. The extinction was measured at 500 nm and the contentof phenylacetohydroxamic acid (▾) or acetohydroxamic acid (♦) wasdetermined by means of the appropriate calibration lines (cf. FIG. 1).Corresponding batches without quiescent cells were incubated in parallelin order to exclude the production of hydroxamic acid by a chemicalreaction. No hydroxamic acid formation could be detected in the controlbatches over the observation period of 30 min.

In a further batch (total volume 4 ml, OD₅₄₆nm=1.0), quiescent cells ofthe MP50 strain were incubated with PAA (100 mM) without hydroxylamine.0.3 ml of the cellular suspension was withdrawn every 5 min, the cellswere removed by centrifugation and the concentration of phenylaceticacid (▪) was determined by means of HPLC.

As shown in FIG. 1, with acetamide and phenylacetamide (PAA) assubstrates in the presence of hydroxylamine, Rhodococcus erythropolisMP50 shows an acyltransferase activity which is markedly higher than thespecific activity of the cells in the hydrolysis of PAA to thecorresponding carboxylic acid.

The specific activities for the formation of phenylacetic acid andphenylacetohydroxamic acid were 0.54 and 7.83 U/mg protein respectively.Acetohydroxamic acid was formed with a specific activity of 2.22 U/mgprotein.

EXAMPLE 2

Since acyltransferase activity could be detected in quiescent cells ofthe MPSO strain in Example 1, the experiments were repeated with crudeextracts from induced cells. Again it was possible to detectacyltransferase activity towards PAA and acetamide in the presence ofhydroxylamine (FIG. 2). The enzymatic hydrolysis of PAA to phenylaceticacid took place with a specific activity of 0.67 U/mg protein. Thespecific activities in the formation of phenylacetohydroxamic acid andacetohydroxami.c acid were 7.27 U/mg and 7.80 U/mg protein respectively,i.e. more than ten times higher than in the hydrolysis of PAA.

The experimental procedure was as follows:

Cells of Rhodococcus erythropolis MP50 were grown with ketoprofenamide(1 mM) as the nitrogen source and harvested in the late exponentialphase. Crude extract was prepared therefrom. 10 μl of the crude extract(6.5 μg of protein) were diluted with 640 μl of Tris/HCl buffer (30 mM,pH 7.5) in an Eppendorf reaction vessel and mixed with 250 μl of afreshly neutralized, aqueous hydroxylamine hydrochloride solution (2 M).The reaction was started by the addition of 100 μl of a methanolicphenylacetamide solution (PAA, 50 mM) or an acetamide solution (50 mM)in Tris/HCl buffer (30 mM, pH 7.5) and took place at room temperature.The total volume was 1 ml. The concentrations of the amide and thehydroxylamine in the reaction were 5 mM and 0.5 M. 100 μl of the batchwere withdrawn every two minutes and mixed with 800 μl of an iron(III)chloride solution acidified with hydrochloric acid. The strongly acidicsolution denatured the protein and stopped the enzymatic reaction.Precipitated protein was centrifuged off and the extinction of thesupernatant was determined by spectrophotometry at 500 nm. Theconcentrations of phenylacetohydroxamic acid (▾) and acetohydroxamicacid (♦) were determined by means of the appropriate calibration lines.To determine the amidase activity, 10 μl of crude extract were dilutedin 0.89 ml of Tris/HCl buffer (30 mM, pH 7.5). The reaction was startedby the addition of 100 μl of methanolic PAA solution. The total volumewas 1 ml and the PAA concentration was 5 mM. 100 μl aliquots of thereaction mixture were pipetted at regular intervals into 10 μl of 1 NHCl, precipitated protein was centrifuged off and the phenylacetic acidconcentration (▪) was determined by means of HPLC.

EXAMPLE 3

An acyltransferase activity could be detected in quiescent: cells and incrude extract of the MPSO strain. It still remained to demonstratewhether the amidase activity and the acyltransferase activity did indeedoriginate from the same enzyme. The formation of hydroxamic acid withamidase purified to homogeneity proved that acyltransferase and amidasewere one and the same enzyme (Table 1).

As shown in Table 1 below, the acyltransferase activity towards2-phenylpropionamide was about three times as high as the amidaseactivity. The formation of acetohydroxamic acid proceeded four times asrapidly as the saponification to acetic acid, and phenylacetohydroxamicacid was even produced nine times as rapidly as phenylacetic acid. Thus,without exception, the amidase from the MP50 strain showed higherspecific activities in the formation of hydroxamic acid than in thehydrolysis of the amides to the carboxylic acids.

TABLE 1 Specific activities in the formation of carboxylic acids andhydroxamic acids from carboxamides by means of the purified amidaseSpec. activity Substrate Product [U/mg protein] acetamide acetic acid0.83 acetamide acetohydroxamic acid 3.54 phenylacetamide phenylaceticacid 4.10 phenylacetamide phenylacetohydroxamic acid 36.612-phenylpropion- 2-phenylpropionic acid 4.50 amide 2-phenylpropion-2-phenylpropionohydroxamic 13.02 amide acid

For determination of the acyltransferase activity, 10 μl of purifiedamidase (2.6 μg of protein) were diluted with 0.64 ml of sodiumphosphate buffer (10 mM, pH 7.5) in an Eppendorf reaction vessel. 0.25ml of a freshly prepared hydroxylamine hydrochloride solution (2 M),neutralized with sodium hydroxide solution, was pipetted in. Thereaction was started by the addition of 0.1 ml of a methanolic stocksolution (50 mM) of the appropriate amide.

Acetamide (50 mM) was dissolved in sodium phosphate buffer (10 mM, pH7.5) and then added. The total volume was 1 ml. The concentrations ofhydroxylamine and the appropriate amide were 0.5 M and 5 mMrespectively. Over a period of 20 min, four 0.1 ml aliquots of thereaction mixture were pipetted into 0.8 ml of iron(III) chloridesolution acidified with hydrochloric acid, and the extinction of thehydroxamic acid complex was measured by photometry at 500 nm. Theconcentration of the hydroxamic acids was determined via the appropriatecalibration lines.

For determination of the amidase activity, the same amount of proteinwas diluted in 0.89 ml of sodium phosphate buffer (10 mM, pH 7.5) andthe appropriate amide (5 mM) was then added. The concentrations of thecarboxylic acids formed were measured by means of HPLC. In the reactionwith acetamide, the amount of ammonium liberated was determined by theindophenol method.

EXAMPLE 4

The fact that acyltransferases, and especially the acyltransferaserecovered from Rhodococcus erythropolis MP50, are active asenantioselective catalysts was demonstrated as follows:

Racemic 2-phenylpropionamide (2-PPA) was chosen as the model substratefor the studies. The purified enzyme was incubated with (R,S)-2-PPA (5mM) in the presence of hydroxylamine (0.5 M), the formation ofhydroxamic acid being monitored both by means of photometry and by meansof ion pair chromatography.

The experiments showed that the formation of hydroxamic acid slowed downdrastically after 50% conversion of the substrate (FIG. 3). Study of theenzymatically formed 2-phenylpropiono-hydroxamic acid (2-PPHA) by meansof a chiral HPLC column showed that only one enantiomer of hydroxamicacid was formed up to 46% conversion of the substrate. The formation of2-phenylpropionic acid could not be observed in the presence ofhydroxylamine. The amidase from the MP50 strain was indeed capable ofconverting racemic 2-PPA enantioselectively to 2-PPHA.

The specific activity at the beginning of the reaction was 9.73 U/mgprotein for the formation of hydroxamic acid, so it was somewhat belowthe maximum attained value of 13.02 U/mg (cf. Table 1) . The marked dropin activity was very clearly observable when 50% of the racemicsubstrate had been converted.

Separation of the enzymatically formed hydroxamic acid by means of achiral HPLC column showed that only one enantiomer appeared up to 46%conversion. The enantiomeric excess was thus greater than 99%.

The experimental procedure was as follows:

10 μl of purified amidase (2.6 μg of protein) were diluted in 0.64 ml ofsodium phosphate buffer (10 mM, pH 7.5) in an Eppendorf reaction vesseland then incubated for 10 min at room temperature with 0.25 ml of afreshly prepared and neutralized hydroxylamine hydrochloride solution (2M). The reaction was started by the addition of 0.1 ml of a methanolicsolution of (R,S)-2-PPA (50 mM stock solution). The total volume was 1ml. The concentration of hydroxylamine was 0.5 M. The initial amideconcentration was 5 mM. 50 μl aliquots of the reaction mixture werepipetted into 5 μl of 1 N HCl at regular intervals. Denatured proteinwas centrifuged off and the concentrations of amide (▾) and hydroxamicacid () were determined by means of ion pair chromatography. Theenantiomeric ratio (◯) of the 2-PPHA was studied with a chiral HPLCcolumn (Chiral-HSA). For this purpose, the 2-PPA and 2-PPHA had to beseparated first through a Grom-Sil 120 TMS-2CP prepacked column.

EXAMPLE 5

The optically pure 2-phenylpropionohydroxamic acid prepared in Example 4was converted to the corresponding 1-phenylethylamine, also opticallypure, by the following procedure:

To remove the excess hydroxylamine, the reaction product obtained inExample 4 was first acidified with 1 N HCl. The pH of the solution wasthen about 0 to 1. Acidification caused the hydroxylamine to decomposeto ammonium and nitrous oxide. This decomposition was assisted bykeeping the reaction mixture at 80° C. in a water bath for 10 min. Whenthe evolution of gas had ceased, the pH was readjusted to about 5 withsodium hydroxide solution.

About 10 times the amount of1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDC) - based on the2-phenylpropionohydroxamic acid—was then added.

The concentration of the 1-phenylethylamine formed was determined bymeans of ion pair chromatography and its enantiomeric excess wasmeasured with the aid of a chiral HPLC column (Crownpak CR(+)).

EXAMPLE 6

A reaction was carried out with the same reactants as those described inExample 5 except that, every time the enzymatic formation of hydroxamicacid was supposed to be stopped, a separate batch was started. This wasdone by diluting 10 μl of the purified amidase (1.7×10⁻³ mg of protein)with 315 μl of sodium phosphate buffer (54 mM, pH 7.5) in an Eppendorfreaction vessel and incubating it with 125 μl of a freshly prepared andneutralized hydroxylamine hydrochloride solution (2 M). The enzymaticreaction was started by the addition of 50 μl of a methanolic solutionof 2-PPA (50 mM). The total volume of each batch was 0.5 ml. Theconcentrations of 2-PPA and hydroxylamine were 5 mM and 0.5 M. 10identical batches were started and were stopped at intervals of 10 minby the addition of 50 μl of 1 N HCl. Precipitated protein was removed bycentrifugation and 50 μl were withdrawn from the batch for HPLCmeasurements to determine the concentrations of 2-PPA and 2-PPHA. Theremaining 500 μl were heated for 10 min at 800C in a water batch inorder to boil off the hydroxylamine. After the samples had cooled toroom temperature, the pH was adjusted to 4.5 by the addition of 29 μl of1 N NaOH. 9.6 mg of EDC (100 mM) were added and the mixture was heatedagain for one hour at 80° C. The concentration of the 1-phenylethylamineformed was determined by means of ion pair chromatography, as in Example3, and its enantiomeric excess was measured with the aid of a chiralHPLC column (Crownpak CR(+)).

EXAMPLE 7

To prepare larger amounts of hydroxamic acid, the initial procedure forrecovering the enzymatically active material is as follows:

A culture medium for growing Rhodococcus erythropolis MP50 is preparedfirst; it has the following composition:

NaHPO₄.12H₂O 14.0 g KH₂PO₄ 2.0 g CaCl₂.2H₂O 0.005 g Fe(III) citrate.7H₂O0.02 g MgSO₄.7H₂O 1.0 g Trace element solution SL6 1.0 ml (after Pfennig& Lipper, 1966) but without EDTA and FeSO₄ Disodium succinate 1.62 gNutrient broth solution 30 ml Phenylacetonitrile 0.18 g H₂O ad 1000 ml

10 ml of this medium were inoculated with 0.1 ml of a liquid culture ofRhodococcus erythropolis MP50 on nutrient. broth and incubated overnightin an Erlenmeyer flask with baffles, at 30° C., on a shaker at 150 rpm.This culture was used to inoculate 100 ml of fresh medium in anErlenmeyer flask with baffles, which was incubated under the sameconditions for 18 to 24 h. This culture in turn was used as an inoculumfor 700 ml of culture medium in a⁻3 1 Erlenmeyer flask with notches. Thecells were harvested by centrifugation in the late exponential growthphase and used, or fast-frozen at −70° C. and stored at 25° C. Celldigestion, crude extract recovery and enzyme purification are carriedout as described in Journal of Bacteriology: B. Hirrlinger, A. Stolz &H. -J. Knackmuss (1996), Purification and Properties of an Amidase fromRhodococcus erythropolis MP50 which Enantioselectively Hydrolyzes2-Arylpropionamides, Journal of Bacteriology 178 (12), pp. 3501-3507.

S-2-Phenylpropionohydroxamic acid was then prepared as follows using theenzymatically active material recovered in this way:

125 U of acyltransferase in the form of whole cells, crude extract orpurified enzyme were mixed, using a magnetic stirrer, with 3.2 1 ofsodium phosphate buffer (10 mM, pH 7.5) in a wide-mouth Schott flaskwith a volume of 10 1. 1.25 1 of a freshly prepared aqueoushydroxylamine hydrochloride solution (2 M), adjusted to pH 7 with sodiumhydroxide solution, were added and the mixture was stirred for 10 min atroom temperature. The enzymatic reaction was started by the addition of3.725 g (0.025 mol.) of (R,S)-phenylpropionamide (dissolved in 500 ml ofmethanol). The course of the reaction was monitored by means of ion pairchromatography. After 120 min, the conversion of the S enantiomer of theamide to the corresponding hydroxamic acid was complete and the cellswere removed by centrifugation, or the crude extract or the purifiedenzyme was denatured by raising the pH to 10 with sodium hydroxidesolution, and then centrifuged off.

To separate the enzymatically formed S-2-phenylpropiano-hydroxamic acidfrom the unconverted R-2-phenylpropionamide, the amide was extractedfrom the alkaline aqueous solution by shaking with ethyl acetate ormethylene chloride. The aqueous solution was then adjusted to pH 2 withconc. HCl and re-extracted with ethyl acetate or methylene chloride.Removal of the organic solvent under vacuum gave 1.9 g (0.012 mol.) ofS-2-phenylpropionohydroxamic acid. The enantiomeric excess, determinedby means of measurements with a chiral HPLC column, was >99%.

EXAMPLE 8

S-2-Phenylpropionohydroxamic acid was prepared as follows using theenzymatically active material prepared as described in Example 7:

125 U of acyltransferase in the form of immobilized whole cells orimmobilized purified enzyme were mixed, using a magnetic stirrer, with3.2 1 of sodium phosphate buffer (10 mM, pH 7.5) in a wide-mouth Schottflask with a volume of 10 1. 1.25 1 of a freshly prepared aqueoushydroxylamine hydrochloride solution (2 M), adjusted to pH 7 with sodiumhydroxide solution, were added and the mixture was stirred for 10 min atroom temperature. The enzymatic reaction was started by the addition of3.725 g (0.02s mol.) of (R,S)-2-phenylpropionamide (dissolved in 500 mlof methanol). The course of the reaction was monitored by means of ionpair chromatography. After 120 min, the conversion of the S enantiomerof the amide to the corresponding hydroxamic acid was complete and theimmobilized cells or the immobilized enzyme were filtered off and storedfor subsequent use in sodium phosphate buffer (10 mM, pH 7.5) at +4° C.

To separate the enzymatically formed S-2-phenylpropiono-hydroxamic acidfrom the unconverted R-2-phenylpropionamide, the aqueous solution wasadjusted to pH with sodium hydroxide solution and the amide wasextracted from the alkaline aqueous solution by shaking with ethylacetate or methylene chloride. The aqueous solution was then adjusted topH 2 with conc. HCl and re-extracted with ethyl acetate or methylenechloride. Removal of the organic solvent under vacuum gave 1.9 g (0.012mol.) of S-2-phenylpropionohydroxamic acid. The enantiomeric excess,determined by means of measurements with a chiral HPLC column, was >99%.

EXAMPLE 9

After O-acylation, the enantiomerically pure hydroxamic acids recoveredby the methods described in Examples 7 to 9 can be converted to theisocyanate by heating or by treatment with bases in aprotic solvents.After the addition of water, the isocyanate forms an unstable carbamicacid, which reacts further to give the primary amine with theelimination of CO₂. As the Lossen rearrangement proceeds with retentionof the configuration at the migrating carbon atom, the products obtainedare optically active primary amines.

EXAMPLE 10

The following procedure was adopted for the production of chiral primaryamines without prior isolation of the hydroxamic acid:

125 U of acyltransferase in the form of whole cells, crude extract orpurified enzyme were mixed, using a magnetic stirrer, with 3.2 1 ofsodium phosphate buffer (10 mM, pH 7.5) in a wide-mouth Schott flaskwith a volume of 10 1. 1.25 1 of a freshly prepared aqueoushydroxylamine hydrochloride solution (2 M), adjusted to pH 7 with sodiumhydroxide solution, were added and the mixture was stirred for 10 min atroom temperature. The enzymatic reaction was started by the addition of3.725 g (0.025 mol.) of (R,S)-2-phenylpropionamide (dissolved in 500 mlof methanol). The course of the reaction was monitored by means of ionpair chromatography. After 120 min, the conversion of the S enantiomerof the amide to the corresponding hydroxamic acid was complete and thecells were removed by centrifugation, or the crude extract or thepurified enzyme was denatured by acidification to pH 1 with conc. HCl,and then centrifuged off.

To remove the excess hydroxylamine, the aqueous solution was heated for30 min at 80° C., with stirring. No further evolution of gas due todecomposing hydroxylamine could then be observed. After the solution hadcooled to room temperature, its pH was adjusted to 4.5 with sodiumhydroxide solution. For derivatization of the hydroxamic acid, 96 g(0.62 mol.) of 1-ethyl-3-(3′-dimethylamino-propyl)carbodiimide (EDC)were added to the aqueous solution, with stirring, and the solution wasthen heated for 2 h at 80° C., with stirring.

To separate the R-2-phenylpropionamide remaining from the enzymaticreaction from the S-1-phenylethylamine formed in the Lossenrearrangement, the aqueous solution was acidified to pH 1 with conc. HCland the amide was extracted by shaking with ethyl acetate or methylenechloride. The aqueous solution was then adjusted to pH 10 with sodiumhydroxide solution and the amine was isolated by shaking with ethylacetate or methylene chloride. Removal of the solvent under vacuum gave1.2 9 (0.096 mol.) of S-1-phenylethylamine with an enantiomeric excessof >99%.

EXAMPLE 11

Analogously to the procedure described in Example 10,S-1-phenylethylamine was prepared as follows, by a Lossen rearrangement,without prior isolation of the corresponding hydroxamic acid:

125 U of acyltransferase in the form of immobilized whole cells orimmobilized purified enzyme were mixed, using a magnetic stirrer, with3.2 1 of sodium phosphate buffer (10 mM, pH 7.5) in a wide-mouth Schottflask with a volume of 10 1. 1.25 1 of a freshly prepared aqueoushydroxylamine hydrochloride solution (2 M), adjusted to pH 7 with sodiumhydroxide solution, were added and the mixture was stirred for 10 min atroom temperature. The enzymatic reaction was started by the addition of3.725 g (0.025 mol.) of (R,S)-2-phenylpropionamide (dissolved in 500 mlof methanol). The course of the reaction was monitored by means of ionpair chromatography. After 120 min, the conversion of the S enantiomerof the amide to the corresponding hydroxamic acid was complete and theimmobilized cells or the immobilized enzyme were filtered off and storedfor subsequent use in sodium phosphate buffer (10 mM, pH 7.5) at +4° C.

To remove the excess hydroxylamine, the aqueous solution was adjusted topH 1 with conc. HCl and heated for 30 min at 80° C., with stirring. Nofurther evolution of gas due to decomposing hydroxylamine could then beobserved. After the solution had cooled to room temperature, its pH wasadjusted to 4.5 with sodium hydroxide solution. For derivatization ofthe hydroxamic acid, 96 g (0.62 mol.) of1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDC) were added to theaqueous solution, with stirring, and the solution was then heated for 2h at 80° C., with stirring.

To separate the R-2-phenylpropionamide remaining from the enzymaticreaction from the S-1-phenylethylamine formed in the Lossenrearrangement, the aqueous solution was acidified to pH 1 with conc. HCland the amide was extracted by shaking with ethyl acetate or methylenechloride. The aqueous solution was then adjusted to pH 10 with sodiumhydroxide solution and the amine was isolated by shaking with ethylacetate or methylene chloride. Removal of the solvent under vacuum gave1.2 g (0.096 mol.) of S-1-phenylethylamine with an enantiomeric excessof >99%.

What is claimed is:
 1. Method of preparing optically active hydroxamic acids of the formula

wherein R¹, R² and R³ are different and are a cyclic or linear, aliphatic or aromatic, substituted or unsubstituted hydrocarbon radical which optionally contain heteroatoms, comprising reacting a racemate of chiral amides, carboxylic acid esters or carboxylic acids of the formula

wherein R¹, R² and R³ are as defined above and X is —NH₂, —OR or —OH, R being any organic radical, with hydroxylamine, NH₂OH, in the presence of an acyltransferase, and then separating the optically active hydroxamic acid (I) formed from the unconverted enantiomer of general formula (II).
 2. Method according to claim 1, wherein the acyltransferase used is an acyltransferase from the microorganisms Mycobacterium smegmatis, Pseudomonas aeruginosa, Arthrobacter, Aspergillus nidulans, Bradyrhizobium japonicum, Methylophilus methylotrophus, Pseudomomas chlororaphis, Pseudomonas fluorescens, Rhodococcus rhodochrous J1 or Rhodococcus erythropolis MP50.
 3. Method according to claim 2, wherein the acyltransferase used is the acyltransferase from Rhodococcus erythropolis MP50.
 4. Method according to claim 2, wherein the acyltransferase is used in the form of a crude extract from the microorganisms.
 5. Method according to claim 2, wherein the acyltransferase is used in purified form.
 6. Method according to claim 1, wherein the reaction is carried out in the presence of a buffer at a pH of 6.5 to 7.5.
 7. Method according to claims 1, wherein the radicals R¹, R² and R³ are selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, cyclohexyl, phenyl, t-butoxyphenyl, naphthyl, 3-benzoylphenyl, amino and hydroxyl.
 8. Method according to claim 7, wherein the compounds of formula (I) used are alkylaminopropionic acid, p-butoxyphenylacetic acid, 2-phenylpropionic acid, 2-methylbutanoic acid, 2-(1-naphthyl)propionic acid, 2-(6-methoxy-2-naphthyl)propionic acid, 2-(3′-benzoylphenyl)propionic acid and their esters or amides.
 9. Method according to claims 1, wherein the compound of formula (I) used is an amide.
 10. Method according to claims 1, wherein the compound of formula (I) is reacted with 2 to 10 times the amount, of hydroxylamine with an acyltransferase activity of 3000 to 5000 units per mol. of substrate.
 11. Method according to claim 10, wherein the compound of formula (I) is reacted with 5 times the amount of hydroxylamine.
 12. Method according to claim 11, wherein the acyltransferase activity is 5000 units per mol. of substrate.
 13. Method according to claim 10, wherein the acyltransferase activity is 5000 units per mol. of substrate.
 14. Method of preparing optically active primary amines of the general formula

wherein R¹, R² and R³ are as defined in claim 1, comprising O-acylating wherein the optically active hydroxamic acid of formula (I), obtained by the method according to claim 1, converting the O-acylated hydroxamicraI acid the corresponding isocyanate by heating or by treatment with bases in aprotic solvents, and reacting the isocyanate with water to give the amine of formula (III) with the elimination of CO₂.
 15. Method of preparing optically active primary amines of the formula

wherein R¹, R² and R³ are as defined in claim 1, comprising reacting, before it is separated from the unconverted enantiomer of formula (II), the optically active hydroxamic acid of formula obtained by the method according to claim 1, with a carbodiimide of the general formula R₄—N═C═N—R₅   (V) wherein R₄ is benzyl, cyclohexyl or ethyl and R₅ is dimethylaminopropyl or morpholinoethyl, and then separating the optically active primary amine of formula (III) formed from the unconverted enantiomer of formula (II).
 16. Method according to claim 15, comprising, before it is reacted with the carbodiimide, acidifying the reaction mixture containing the optically active hydroxamic acid (I) and the unconverted enantiomer (II) to a pH of 0 to 2, then heating the reaction mixture to a temperature of 40° C. to 100° C., wherein the reaction with the carbodiimide is then carried out at a pH of 4 to
 6. 17. Method according to claim 16, wherein the reaction mixture containing the optically active hydroxamic acid (I) and the unconverted enatiomer (II) is acidified to a pH of
 1. 18. Method according to claim 17, wherein the reaction mixture is heated to a temperature of 80° C.
 19. Method according to claim 17, wherein the reaction with the carbodiimide is carried out at a pH of 5.0.
 20. Method according to claim 16, wherein the reaction mixture is heated to a temperature of 80° C.
 21. Method according to claim 20, wherein the reaction with the carbodiimide is carried out at a pH of 5.0.
 22. Method according to claim 16, wherein the reaction with the carbodiimide is carried out at a pH of 5.0.
 23. Method according to claim 15, wherein the carbodiimide used is 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide. 